Azanitrile Inhibitors of the SmCB1 Protease Target Are Lethal to Schistosoma mansoni: Structural and Mechanistic Insights into Chemotype Reactivity

Article abstract of DOI:10.1021/acsinfecdis.0c00644

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E-to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

Full text of DOI:10.1021/acsinfecdis.0c00644

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Article
Azanitrile Inhibitors of the SmCB1 Protease Target Are Lethal to
Schistosoma mansoni: Structural and Mechanistic Insights into
Chemotype Reactivity
‡
‡
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́ ̌ ́
Adela Jílkova, Martin Horn, Jindrich Fanfrlík, Jim Kuppers, Petr Pachl, Pavlína Rezacova,
̈
́
́
̌
̌
́
̌
́
́
Martin Lepsík, Pavla Fajtova, Petra Rubesova, Marta Chanova, Conor R. Caffrey, Michael Gu
and Michael Mares*
̈
tschow,*
̌
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ABSTRACT: Azapeptide nitriles are postulated to reversibly covalently react
with the active-site cysteine residue of cysteine proteases and form
isothiosemicarbazide adducts. We investigated the interaction of azadipeptide
nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a
pathogen that causes the global neglected disease schistosomiasis. Azadipeptide
nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We
determined the crystal structure of SmCB1 in complex with an azadipeptide
nitrile and analyzed the reaction mechanism using quantum chemical
calculations. The data demonstrate that azadipeptide nitriles, in contrast to
their carba counterparts, undergo a change from E- to Z-configuration upon
binding, which gives rise to a highly favorable energy profile of noncovalent and
covalent complex formation. Finally, azadipeptide nitriles were considerably
more lethal than their carba analogs against the schistosome pathogen in
culture, supporting the further development of this chemotype as a treatment
for schistosomiasis.
KEYWORDS: azapeptide inhibitors, cysteine proteases, protein structures, structure−activity relationships, schistosomiasis
(bilharzia), a chronic disease that infects over 200 million
zapeptides, peptides in which the CαH of at least one
Aamino acid has been replaced with nitrogen, have people in tropical and subtropical areas.21 As treatment and
control of schistosomiasis rely on just one drug, praziquantel,
there is impetus to identify new antischistosomals.22−25 The
cysteine protease SmCB1 is a central digestive enzyme of the
parasite and has been validated as a chemotherapeutic target
for the cure of schistosomiasis.20,26 Previously, we determined
the crystal structure of SmCB1 as a footing for rational drug
development.20,27,28
In this study, we compared the functional properties of
dipeptides with azanitrile and carbanitrile warheads and found
azanitriles to be superior. The azadipeptide nitriles were potent
inhibitors of SmCB1 with antischistosomal activity and, thus,
represent a new class of potential drug leads. We present the
first crystallographic analysis of a protease−azanitrile inhibitor
complex and quantum chemical calculations to provide
emerged as particularly important peptidomimetic structures
(Figure 1). Compared to their parent carbapeptide analogs,
bioactive azapeptides can possess improved potency and target
selectivity as well as superior pharmacokinetics.1−5 Azadipep-
tide nitriles were introduced as a class of efficient inhibitors of
human cysteine cathepsins.6−9 This chemotype supported the
successful development of activity-based probes, modified for
organelle-specific delivery to lysosomal cysteine proteases and
applied as PET-imaging agents for tumor-associated cathepsin
activity.10−12 These reports have highlighted that azadipeptide
nitriles can enrich the portfolio of inhibitors of cysteine
proteases suitable as activity-based probes13,14 and potential
therapeutics against parasitic and protozoal infections.15−18
Similar to the well-established dipeptide nitriles, azadipeptide
nitriles are thought to undergo a covalent, reversible
interaction with the target proteases by forming a stabilized
isothiosemicarbazide adduct (Figure 1).6−12
Received: September 10, 2020
Here, we investigated the interaction of aza- and
carbadipeptide nitriles with cathepsin B1 (SmCB1) from the
human blood fluke, Schistosoma mansoni.19,20 Trematode
flatworms of the genus Schistosoma cause schistosomiasis
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Table 1. Pairs of Peptidomimetics with Aza- or Carbanitrile
Warheads and Their Inhibition Potency against SmCB1
Figure 1. Carba- and azadipeptide nitriles and their reaction with
cysteine proteases. Isoelectronic CαH/N exchange in the warhead
(cyan/magenta) of dipeptide nitriles (left) leads to azadipeptide
nitriles (right). Two carbohydrazide nitrogens in azanitriles need to
be alkylated to circumvent spontaneous heterocyclization.6 R1 and R2
are substituents in amino acid residues at the P1 and P2 positions
(binding in the enzyme subsites S1 and S2), respectively; PG is a
protecting group. The depicted azadipeptide bears an aza-alanine
nitrile at the P1 position. Reactive warheads of both chemotypes form
a covalent, reversible bond with the thiol of the catalytic cysteine
residue (orange) of papain-family cysteine proteases (represented by
SmCB1).
mechanistic insight into the phenomenon of azanitrile warhead
reactivity.
RESULTS AND DISCUSSION
■
SAR Analysis of Aza- and Carbadipeptide Nitriles
Reveals a High Potency of SmCB1 Inhibitors with an
Azanitrile Warhead. We have evaluated a set of 18
azadipeptide and 50 carbadipeptide nitriles in vitro as potential
inhibitors of the SmCB1 protease. The compound scaffold is
defined by positions P3 through P1 (Schechter and Berger
nomenclature),29 and the substitutions were selected to
provide a high diversity in all positions. The compounds
were screened against recombinant SmCB1, and their Ki values
were determined using a kinetic inhibition assay with the
fluorogenic substrate Cbz-Phe-Arg-AMC. The data for seven
representative pairs of both chemotypes are shown in Table 1.
Analogs with the azanitrile warhead (1a−7a) were more
potent than those with the carbanitrile warhead (1c−7c). This
general trend was confirmed for the entire set of test
compounds (Tables S1 and S2). Most of the aza compounds
were effective in nanomolar concentrations with slow-binding
kinetics, whereas only 10% of the carba compounds had Ki
values <1 μM and had fast-binding behavior with linear
progress curves (Figure 2), a typical feature of peptidic
(carba)nitrile inhibitors of cysteine proteases.9,30,31 Using the
example of the Cbz-capped phenylalanine pair (2a and 2c),
additional variations in the carbanitrile warhead by the
stepwise introduction of methyl groups at the amino-
acetonitrile unit (i.e., the incorporation of alanine, sarcosine,
or N-methylalanine-derived nitrile building blocks in 8c−10c)
did not improve the inhibition of SmCB1 (Table 2).
a
The Ki values were determined using a kinetic activity assay with the
fluorogenic peptide substrate Cbz-Phe-Arg-AMC.
active. Larger hydrophobic and aromatic residues at the P2
position strengthen binding to the protease (1a versus 2a,
11a). Also, modification of the α-nitrogen substituent in the
aza-amino nitrile unit of 2a by extended hydrophobic P1
substituents could improve inhibition (2a versus 8a, 9a). In the
P3 position, the introduction of a benzyl urea moiety as
present in 3a markedly increased the inhibitory potency. A
comparison of P3 triaryl compounds, each with leucine in the
P2 position (4a, 5a, 17a), showed that the amide linkage and
the associated triaryl group (5a) was preferred over the urea
and methylurea linkages (4a and 17a, respectively). The amide
bond contributes to the binding affinity because its
methylation decreased potency 300-fold (5a versus 6a).
When the entire structure of 5a was maintained but leucine
was replaced with a less appropriate P2 amino acid, i.e.,
homocycloleucine in 7a, a 40-fold decrease in potency was
measured. Also, we analyzed the second-order rate constants of
azadipeptide nitriles (k2nd, Table S1). The k2nd values of the
five most potent compounds with single-digit nM Ki values
(from 4.6 to 6.2 nM) were particularly high for 3a, 5a, and 11a
Table 3 shows the SAR analysis of the P1 to P3 positions of
the azanitrile scaffold. The presence of the azanitrile warhead
was not sufficient for strong potency, as compound 1a, which
lacks a residue side chain for the P2−S2 interaction, was not
B
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substituents in 8a and 9a (R1 = Bn−CH2− and CH3−
(CH2)4−, respectively).
Phenotypic Effects of Azanitriles on Schistosoma
mansoni. A panel of 35 azanitrile and carbanitrile inhibitors
that were effective against SmCB1 (Tables 1−3 and S2) was
phenotypically screened in vitro at 10 μM against S. mansoni
newly transformed schistosomula (NTS), the postinvasive
stage of the parasite that feeds on host blood. The resulting
phenotypic responses were graded from 0 through 4, i.e., from
the least to the most severe. Figure 3 compares the severity
scores for the azanitrile and carbanitrile analog pairs (for
complete phenotyping data for the panel, see Table S3). We
found that both inhibitor chemotypes substantially differed in
their bioactivities.
The azanitrile inhibitors caused rapid and severe phenotypes
(scores of 3 and 4) except for 1a, which was ineffective against
NTS and SmCB1. In contrast, the incubation of NTS with the
carbanitrile analogs resulted in more occasional and lower
severity scores. The correlation between the severity scores
generated by the azanitriles against the parasite and their
potency of inhibition of SmCB1 was highly significant (Table
S3, Spearman correlation test with nonzero value coefficients,
20 000 permutations, p < 0.001). The most inhibitory
azanitriles (with Ki values in the single-digit nM range) were
also tested for their cytotoxicity against two human cell lines.
They displayed low cytotoxicity at the same concentrations
used in the NTS assay (Table S4), indicating that the observed
phenotypic changes were specific to the parasite. In conclusion,
azadipeptide nitriles that target SmCB1 were demonstrated to
be effective antischistosomal compounds.
Crystallographic Analysis Identifies the Binding
Mode of Azanitriles to SmCB1. The crystal structure of
SmCB1 in complex with the single-digit nanomolar inhibitor
3a was determined at a resolution of 1.3 Å (PDB ID: 6YI7;
Table S5). The binding mode of 3a in the active site is
presented in Figure 4. The inhibitor’s P1 to P3 residues occupy
the S1 to S3 binding subsites of SmCB1. Through the
nucleophilic attack of the thiol group of the catalytic Cys100
residue, an isothiosemicarbazide adduct is formed that
incorporates the CAB atom of the nitrile moiety (Figures 4D
and S1). The NAA atom of the nitrile moiety is stabilized by
two hydrogen bonds to the backbone amide of Cys100 and the
side chain amide of Gln94 (Figure 4D). Analogous interactions
were observed in the structure of rat cathepsin B complexed
with a carbanitrile inhibitor.32
Figure 2. Different binding kinetics of SmCB1 inhibitors with
azanitrile and carbanitrile warheads represented by 2a and 2c,
respectively. Progress curves show the hydrolysis of the fluorogenic
substrate Cbz-Phe-Arg-AMC by SmCB1 in the presence of increasing
inhibitor concentrations. (A) The azanitrile exhibited a time-
dependent inhibition characterized by nonlinear progress curves
typical of slow-binding kinetics. (B) Linear progress curves obtained
for the carbanitrile are characteristic of fast-binding inhibitors. In
dose−response plots, the derived steady-state reaction velocities were
plotted against inhibitor concentration, and the inhibition constants
Ki were obtained. In the kobs versus [I] plot, the first-order rate
constants kobs from the time-dependent progress curves were plotted
against inhibitor concentrations to show a linear dependence. For
details on data fitting and kinetic parameters, see the Methods.
Table 2. Effect of Variations in the Carbanitrile Warhead on
SmCB1 Inhibition
Azadipeptides are atropochiral molecules due to the
restricted rotation around the methylated N−N axis. The
induction of chirality was demonstrated by methylation of the
hydrazine fragment in model azapeptides, which led to the E-
configuration of the respective CO−N bond and, hence, to
atropisomerism.33 However, E-configured peptidomimetic
ligands should possess a weaker affinity for the active site of
a protease than the Z-configured isomers. This is also
supported by data for cathepsin inhibition involving E- and
Z-locked azadipeptide nitriles.34 The configuration of non-
locked azadipeptide nitriles bound to the protease active site is
so far unknown. Noteworthy, in this study, we were able to
demonstrate the Z-configuration of the C(O)−NAE(NAC)
fragment for the enzyme-bound prototypical inhibitor 3a
(Figure 4).
a
The Ki values were determined using a kinetic activity assay with the
fluorogenic peptide substrate Cbz-Phe-Arg-AMC.
(20.4, 29.0, and 32.7 × 103 M−1 s−1, respectively) and
somewhat lower for 8a and 9a (5.2 and 2.1 × 103 M−1 s−1),
indicating a slower association of the enzyme−inhibitor
complex, presumably due to the presence of the large P1
Insights were also gained into the binding with the S2 and
S3 subsites. At the P2 position, 3a contains a leucine
substituent making nonpolar interactions with residues
C
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Table 3. Inhibition of SmCB1 and Antischistosomal Activity of Azadipeptide Nitriles
a
b
Compounds 1a to 7a are also presented in Table 1. The abbreviations used are as follows:
c
d
The Ki values were determined using a kinetic activity assay with the fluorogenic peptide substrate Cbz-Phe-Arg-AMC. Phenotypic changes in
the parasite in the presence of 10 μM inhibitor are indicated for 24 and 48 h (see Table S3) and converted to a severity score on a scale from 0 (no
e
f
effect) to 4 (severe). 2a is listed in both P1 and P2 sections. N-Methylated leucine.
Ala271 and Leu146 of SmCB1 (Figure 4C). Residue Glu316 in
the S2 subsite has been shown to rotate out of the binding
pocket to avoid steric clash with bulky ligand substituents.20 In
the SmCB1−3a complex, the flexibility of Glu316 was
demonstrated by its dual conformation with one of the
conformations making contact with the P2 leucine. The P3
position of the inhibitor is formed by a benzylaminocarbonyl
group. The lower value of the electron density of this
substituent suggests its static or dynamic disorder (Figure
S2). In the crystallographic model, one conformation of the
benzylaminocarbonyl moiety was modeled into the electron
density map, representing the major position. Nevertheless, it
is obvious that the P3 substituent can acquire several
alternative positions within the S3 subsite, which is rather
wide and less engaged in ligand binding.20
Quantum Chemical Calculations Demonstrate the
Highly Favorable Energetics of the Azanitrile Reaction
Mechanism. Computational approaches based on quantum
mechanics have proven powerful in exploring catalytic
mechanisms of cysteine proteases and designing their covalent
inhibitors.20,35−39 In the present study, quantum chemical
calculations were employed to analyze the “free” energy profile
of the binding reaction of 3a to SmCB1, proceeding via a
noncovalent intermediate to the final covalent complex (Figure
5A; “free” energies refer to the sums of gas phase energies and
solvation free energies). We observed different configurations
of the planar amide group C(O)−N(N) at the warhead
(Figure 5B). The bound 3a adopts the Z-configuration as seen
in the SmCB1−3a crystallographic complex; however, the
modeling of unbound 3a reveals that the E-configuration is
more stable in solvent (by 1.9 kcal mol−1) (Table S6 and
D
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goes a conformational change upon binding to the enzyme.
The activation energy for the E- to Z-conversion calculated
using the implicit solvent model was approximately 15 kcal
mol−1 (Table S6). This rotational barrier is a possible
explanation for the slow binding of azanitriles to SmCB1
observed in the inhibition kinetics (Figure 2). On the contrary,
for the carbanitrile, 3c, the Z-configuration of the C(O)−N(C)
moiety was more stable than the E-configuration for both the
modeled SmCB1−3c complex and the uncomplexed 3c (by
3.1 and 2.0 kcal mol−1, respectively) (Figure S3 and the
Supplementary Results). This agrees with the Z-configuration
reported for various protease-bound and free carbanitriles.32,33
The fact that no conformational change is required for the
interaction of 3c with the enzyme determines the fast-binding
kinetics of carbanitriles to SmCB1 (Figure 2).
We next investigated the intermediate noncovalent com-
plexes of 3a and 3c with SmCB1. In optimized noncovalent
models, the catalytic residue His270 is positively charged, and
the thiolate sulfur atom of Cys100 is separated by about 3.2 Å
from the CAB atom of the inhibitor (Figure 5C). The formation
of the noncovalent complex is energetically favorable for both
inhibitors with a higher “free” energy gain for 3a (Figure 5A,
Table S6). The stronger noncovalent interaction of 3a might
be the result of a distinct electrostatic pattern of the azanitrile
warhead (Figure S4 and the Supplementary Results).
Conversion to the covalent complex is associated with the
formation of a covalent bond between the S atom of Cys100
and CAB atom of the inhibitor and a concomitant water-
mediated proton transfer from His270 to the NAA atom of the
nitrile group (Figure 5C). The transformation proceeds via a
transition state with the S···CAB distance of about 2.3 Å and a
proton localized on H3O+ (crystallographic water molecule
628). The transition barrier is 11.1 and 19.2 kcal mol−1 for 3a
Figure 3. Correlation of the antischistosomal activity with the
inhibition of SmCB1 by azanitrile and carbanitrile analogs. (A) Pairs
of analogs with azanitrile and carbanitrile warheads were phenotypi-
cally screened against S. mansoni newly transformed schistosomula
(NTS) and the data arising was compared to those for the inhibition
of SmCB1. Phenotypic changes in the parasite (Table S3) were
observed every day for 4 days in the presence of 10 μM inhibitor.
Changes were converted to a severity score on a scale from 0 (no
effect) to 4 (severe; red heat map). Ki values for SmCB1 inhibition
(from Table 1) are shown in the blue heat map. (B) An example of an
inhibitor-induced degenerated phenotype in NTS of S. mansoni versus
untreated controls (for details, see Table S3).
Figure S3). This is in line with the preferred E-conformation
determined experimentally for other free azanitriles and
trisubstituted hydrazides in general.33,34,40,41 Thus, 3a under-
Figure 4. Binding mode of the azadipeptide nitrile inhibitor 3a in the SmCB1 active site. (A) Overall crystal structure of the SmCB1−3a complex
in surface (enzyme) and stick (inhibitor) representation. In the SmCB1 active site (boxed), the catalytic residues Cys100 (yellow) and His270
(pink) and major subsite residues (cyan) are highlighted. (B) Chemical structure of 3a forming a covalent bond with the S atom of the catalytic
Cys100. The azanitrile warhead is boxed in gray, and atom labeling is indicated (hydrogen atoms are omitted). (C) Zoomed view of (A) showing
the active site residues that form nonpolar interactions (green) with 3a (in stick representation with carbon atoms in magenta); the catalytic
residues are also indicated. (D) The P1 to P3 positions of the inhibitor bind the corresponding S1 to S3 subsites of the SmCB1 active site. Dashed
lines indicate hydrogen bonds formed between SmCB1 residues (gray) and 3a (magenta); heteroatoms have standard color coding (O, red; N,
blue; S, yellow). Coordinates are deposited under PDB code 6YI7.
E
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Figure 5. Computational analysis of the binding reaction of azanitrile and carbanitrile inhibitors to the active site of SmCB1. (A) The “free” energy
profile of the binding of azanitrile 3a and carbanitrile 3c was determined using quantum chemical calculations. Individual states along the reaction
pathway (indicated by numbers) are defined by their relative “free” energies (Table S6). (B) The unbound azanitrile inhibitor has the E-
configuration in solution (with minimum “free” energy) and undergoes a conformational change to the Z-configuration that was also demonstrated
crystallographically in the SmCB1−3a complex. (C) Modeled states upon binding of the azanitrile inhibitor to the active site include an initial
noncovalent complex (4), a transition state with proton transfer from His270 to H2O (5), and a final covalent complex after proton transfer to the
nitrile group (6). The distance (sulfur−carbon) of the catalytic Cys100 and the inhibitor’s CAB atom is 3.2, 2.3, and 1.8 Å, respectively.
and 3c, respectively, and the transition state has lower “free”
energy than the initial separated reactants (Table S6). The
final covalent complexes with a S−CAB separation of 1.8 Å are
more stable by 9.2 and 5.4 kcal mol−1 for 3a and 3c,
respectively, compared to the noncovalent complexes, thus
indicating that the “free” energy gain is higher for 3a also in the
last step of the reaction (Table S6).
mol−1 compared to its carbanitrile analog (Table S6), which
corresponds to their inhibitory potencies differing by several
orders of magnitude (Table 1). The computational analysis
showed that the carbanitrile inhibitor has the Z-configuration
both in solution and in covalent complex with SmCB1,
whereas the azanitrile inhibitor undergoes the E- to Z-
transformation. Further, the Z-configuration of the azanitrile
in the SmCB1 noncovalent complex is more stable than the E-
configuration (by 3.5 kcal mol−1, Supplementary Results),
To conclude, the azanitrile 3a has more favorable
thermodynamics of its reaction with SmCB1 by 10.2 kcal
F
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a
Scheme 1. Synthesis of Compound 3c
a
Reagents and conditions: (a) (1) ClCO2i-Bu, NMM, THF, −25 °C and (2) H2NCH2CN × H2SO4, NaOH, H2O, THF, −25 °C to rt, 2 h; (b)
TFA, CH2Cl2, rt, 2 h; (c) benzyl isocyanate, Et3N, CH2Cl2, 0 °C for 15 min to rt for 16 h.
suggesting that azanitrile binding involves a “configurational
selection”42 determined by the topology of the SmCB1 active
site. The linear dependence of the kobs values on the inhibitor
concentrations (Figure 2) indicates a one-step kinetic reaction
mechanism of the slow-binding azanitrile inhibitor,43 which is,
however, composed of several distinct mechanistic events in
the physicochemical reaction scheme. On the basis of
computational analysis, E- to Z-conversion is the kinetic
controlling step, while the covalent bond formation is not
supposed to be attributed to the slow binding because the
transition barrier TS2 of azanitrile is even lower than that of the
fast-binding carbanitrile. The slow binding observed for
azanitriles is a known kinetic feature of several other
chemotypes of covalent inhibitors of cysteine proteases.35,44−46
METHODS
■
Synthesis of Inhibitors. General Conditions. Except for
compound 3c, the inhibitors used in this study have been
synthesized as described.6−8,47 Thin-layer chromatography was
carried out on Merck (Darmstadt, Germany) aluminum sheets,
silica gel 60 F254. Detection was performed with a UV light at
254 nm. Preparative column chromatography was performed
on Merck silica gel (0.063−0.200 mm, 60 Å). Melting points
were determined on a Buchi (Essen, Germany) 510 oil bath
̈
1
apparatus. H NMR (500 MHz) and 13C NMR (125 MHz)
spectra were recorded on a Bruker Avance DRX 500
spectrometer and H NMR (600 MHz) and 13C NMR (150
1
MHz) spectra, on a Bruker Avance III 600 NMR spectrometer.
Chemical shifts δ are given in ppm referring to the signal
center using the solvent peaks for reference: DMSO-d6 2.49/
39.7 ppm. LC-MS analyses were carried out on an API2000
(Applied Biosystems, Darmstadt, Germany) mass spectrometer
coupled to an Agilent (Santa Clara, CA, USA) 1100 LC system
using a Phenomenex Luna C18 column (Phenomenex,
Aschaffenburg, Germany; 50 × 2.0 mm, particle size 3 μm).
The purity of the compounds was determined using the diode
array detector (DAD) of the LC-MS instrument between 196
and 400 nm. HRMS spectra were recorded on a microTOF-Q
CONCLUSIONS
■
We investigated the inhibition potency of dipeptidomimetics
with azanitrile and carbanitrile warheads against the cysteine
protease SmCB1, a drug target from the human blood fluke
S. mansoni. A screen of 68 compounds, including analog pairs
of both chemotypes, demonstrated the nanomolar inhibition
potency of the azanitriles and their general superior potency
over their carbanitrile counterparts. Furthermore, the azani-
triles were more quickly lethal to the schistosome parasite in
vitro and, accordingly, represent a new class of compounds for
the development of schistosomiasis drugs. The study provides
a platform for further improvement of the azanitrile scaffold
based on, for example, the introduction of a P3 triaryl moiety
that is connected via an amide bond to a hydrophobic P2
amino acid and that proved particularly advantageous for
SmCB1 inhibition. Further research will also address the
selectivity of azanitrile inhibitors that are, in general, highly
potent against human cysteine cathepsins with endopeptidase
activity.6,7
̈
(Bruker, Koln, Germany) mass spectrometer connected to a
Dionex (Thermo Scientific, Braunschweig, Germany) Ultimate
3000 LC via an ESI interface using a Nucleodur C18 Gravity
column (50 × 2.0 mm I.D., 3 μm, Macherey-Nagel, Duren,
̈
Germany). The compounds were of at least 95% purity. All
compounds passed the PAINS filter using a false positive
remover.48
(S)-tert-Butyl 1-(Cyanomethylamino)-4-methyl-1-oxopen-
tan-2-ylcarbamate (II).
The atomic-level azanitrile−target interaction has not been
elucidated up to now. In this study, we solved the first crystal
structure of SmCB1 in complex with an azadipeptide nitrile
and structurally analyzed the azanitrile-binding mode. By then
analyzing the quantum chemical “free” energy profiles of the
azanitrile- and carbanitrile-binding reactions with SmCB1, we
demonstrated that the azanitriles alone undergo an E- to Z-
conformational change upon binding to the enzyme, which we
propose as an explanation for the different inhibition kinetic
behavior of both chemotypes. Furthermore, azanitriles have a
significantly higher “free” energy gain in three consecutive
steps along the reaction coordinate, including formation of the
initial noncovalent inhibitor−enzyme complex, the transition
state, and the final covalent complex with the inhibitor
warhead linked to the enzyme catalytic site. The data on the
distinct conformational dynamics and binding energetics of the
azanitrile warhead provide a mechanistic insight into the
reactivity of azanitrile peptidomimetics.
(S)-2-(tert-Butoxycarbonylamino)-4-methylpentanoic acid hy-
drate (I, 5.39 g, 21.6 mmol) was dissolved in anhydrous THF
(30 mL) and cooled to −25 °C. N-Methylmorpholine (2.61
mL, 2.41 g, 23.8 mmol) and isobutyl chloroformate (3.10 mL,
3.25 g, 23.8 mmol) were consecutively added to the stirred
solution. Aminoacetonitrile monosulfate (6.65 g, 43.1 mmol)
was suspended in H2O (3 mL). The resulting suspension was
treated with 5 N NaOH (17.0 mL) under ice-cooling and
added to the reaction mixture when the precipitation of N-
methylmorpholine hydrochloride occurred. It was allowed to
warm to room temperature within 0.5 h and stirred for a
further 1.5 h. After evaporation of the solvent, the resulting
aqueous residue was extracted with EtOAc (3 × 30 mL). The
combined organic layer was washed with aq. 10% KHSO4 (30
G
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mL), H2O (30 mL), a sat. aq. NaHCO3 solution (30 mL),
H2O (30 mL), and brine (30 mL). The solvent was dried over
Na2SO4, filtered, and evaporated to dryness. The crude residue
was purified by recrystallization from EtOAc/n-hexane to
obtain II as a white solid (4.01 g, 69%); mp 118−120 °C (lit.49
mp 114−116 °C) (Scheme 1).
157.76 (NH−CO−NH), 174.07 (CH−CO−NH). LC-MS
(ESI) (90% H2O to 100% MeOH in 10 min, then 100%
MeOH for 10 min, DAD 210−400 nm), 99% purity, m/z =
302.9 ([M + H]+). HRMS, calcd. for C16H22N4O2: [M + H]+
m/z 303.1814; found: 303.1816.
Production of Recombinant SmCB1. A nonglycosylated
mutant of the SmCB1 zymogen (Uniprot accession Q8MNY2)
was expressed using the pPICZαA vector in the yeast Pichia
pastoris, activated by S. mansoni legumain and purified as
described previously.27,50 All purification steps were performed
under reducing conditions in the presence of 2 mM
dithiothreitol and 1 mM EDTA under an argon atmosphere
to prevent the active site cysteine residue from oxidation.
Preparation of the SmCB1−Inhibitor Complex. The
freshly activated SmCB1 (0.5 mg mL−1) was incubated with a
4-fold molar excess of the inhibitor 3a in 10 mM sodium
acetate, pH 5.5, containing 20 mM cysteine and 1 mM EDTA,
for 3 h at room temperature under an argon atmosphere. The
enzyme inhibition was monitored using a kinetic assay with the
fluorogenic substrate Cbz-Phe-Arg-AMC.20 The complex was
buffer-exchanged into 5 mM sodium acetate, pH 5.5, and
concentrated to the final concentration of 4 mg mL−1 using
Amicon Ultracel-10k centrifugal units (Millipore, Burlington,
USA); the inhibitor 3a was maintained in a 4-fold molar excess
to SmCB1 in the mixture.
1H NMR (600 MHz, DMSO-d6) δ 0.83 (d, 3J = 6.5 Hz, 3H)
and 0.86 (d, 3J = 6.6 Hz, 3H, CH(CH3)2), 1.33−1.39 (m, 1H,
2
CH−CHH), 1.36 (s, 9H, C(CH3)3), 1.43 (ddd, J = 13.6 Hz,
3J = 10.1 Hz, 3J = 5.2 Hz, 1H, CH−CHH), 1.52−1.63 (m, 1H,
CH(CH3)2), 3.95 (td, 3J = 9.1 Hz, 3J = 4.8 Hz, 1H, NH−CH),
3
4.08−4.12 (m, 2H, CH2−CN), 6.96 (d, J = 8.2 Hz, 1H,
3
O−CO−NH), 8.52 (t, J = 5.7 Hz, 1H, CH−CO−NH). 13C
NMR (150 MHz, DMSO-d6) δ 21.56, 23.06 (CH(CH3)2),
24.41 (CH(CH3)2), 27.25 (NH−CH2), 28.35 (C(CH3)3),
40.58 (CH−CH2), 52.65 (NH−CH), 78.27 (C(CH3)3),
117.74 (CN), 155.55 (O−CO−NH), 173.49 (CH−CO−
NH). LC-MS (ESI) (90% H2O to 100% MeOH in 10 min,
then 100% MeOH for 10 min, DAD 196−200 nm), 100%
purity, m/z = 270.0 ([M + H]+), 287.0 ([M + NH4]+).
(S)-2-(3-Benzylureido)-N-(cyanomethyl)-4-methylpenta-
namide (3c).
Protein Crystallization and Data Collection. Crystals of
the SmCB1−3a complex were obtained by vapor diffusion in a
hanging drop. The drop consisting of 1 μL of protein solution
and 1 μL of reservoir solution was equilibrated over 1 mL of
reservoir solution at 20 °C. The reservoir solution contained
180 mM ammonium acetate, 80 mM sodium citrate, and 27%
PEG 1500, pH 6.1. The protein concentration of the stock
solution of the complex was 4 mg mL−1 (in 5 mM sodium
acetate, pH 5.5). The obtained needle-shaped crystal was flash-
cooled by plunging into liquid nitrogen without cryoprotec-
tion. Diffraction data were collected at 100 K on the beamline
MX14.1 operated by the Helmholtz-Zentrum Berlin at the
BESSY II electron storage ring (Berlin-Adlershof, Germany).51
Diffraction data were processed using the XDS suite of
programs.52 Crystal parameters and data collection statistics
are given in Table S5.
Crystal Structure Determination. The SmCB1−3a
complex crystallized in the orthorhombic space group
P212121 containing one molecule in the asymmetric unit and
a solvent content of ∼41% (Table S5). The structure of the
SmCB1−3a complex was determined by molecular replace-
ment with the program Molrep53 from the CCP4 package54
using the structure of the mature SmCB1 (PDB code: 3S3Q)20
as the search model. Model refinement was carried out using
the program REFMAC 5.2 from the CCP4 package,54
interspersed with manual adjustments using Coot.55 The
structure was refined using data to a resolution of 1.3 Å. The
final crystallographic model contains residues 70−323 (the
SmCB1 zymogen numbering is used throughout the paper).
Anisotropic refinement of all atomic displacement parameters
(ADPs; B-factors) was included in the refinement protocol.
The geometric restraints for ligand 3a were constructed by the
program Libcheck54 using 3a optimized by the DFT-D3/
B3LYP/DZVP method combined with the COSMO56 implicit
solvent model. The optimization was performed by the
Turbomole7.057 and Cuby458 programs. The 3a molecule
was modeled with an occupancy factor of 1 into generally well-
defined electron density. The final refinement statistics are
(S)-tert-Butyl 1-(cyanomethylamino)-4-methyl-1-oxopentan-2-
ylcarbamate (II, 2.02 g, 7.50 mmol) was dissolved in
anhydrous CH2Cl2 (200 mL) and treated with TFA (50
mL). The reaction mixture was stirred at room temperature for
2 h. The solvent was then evaporated, and the residue was
diluted with CH2Cl2 (4 × 50 mL) and evaporated to remove
the excess TFA. The crude product was dissolved in anhydrous
CH2Cl2 (30 mL) and cooled to 0 °C. Subsequently, benzyl
isocyanate (2.01 mL, 2.18 g, 16.3 mmol) was added, followed
by triethylamine (5.15 mL, 3.76 g, 37.1 mmol). Stirring at 0 °C
was prolonged for 15 min, and it then continued at room
temperature overnight. After evaporation of the solvent, the
residue was suspended in H2O (50 mL) and extracted with
EtOAc (3 × 75 mL). The organic layer was washed with aq.
10% KHSO4 (50 mL), H2O (50 mL), a sat. aq. NaHCO3
solution (50 mL), H2O (50 mL), and brine (50 mL), dried
over Na2SO4, filtered, and evaporated to dryness. The crude
residue was purified by preparative column chromatography
using a gradient of petroleum ether/EtOAc (1:1) to EtOAc
(100%), followed by recrystallization from EtOAc of the
product containing fractions to give a white solid (0.34 g,
15%); mp 192−193 °C (Scheme 1).
1H NMR (500 MHz, DMSO-d6) δ 0.85 (d, 3J = 6.6 Hz, 3H)
and 0.88 (d, 3J = 6.7 Hz, 3H, CH(CH3)2), 1.33−1.45 (m, 2H,
CH−CH2), 1.53−1.63 (m, 1H, CH(CH3)2), 4.07−4.11 (m,
2H, CH2−CN), 4.18−4.22 (m, 2H, CH2−NH−CO−NH),
3
3
6.17 (d, J = 8.4 Hz, 1H, NH−CH), 6.42 (t, J = 6.0 Hz, 1H,
CH2−NH−CO−NH), 7.17−7.25 (m, 3H, 2H, 4H), 7.26−
3
7.33 (m, 2H, 3H), 8.66 (t, J = 5.6 Hz, 1H, CH−CO−NH).
13C NMR (125 MHz, DMSO-d6) δ 21.96, 23.09 (CH(CH3)2),
24.42 (CH(CH3)2), 27.19 (NH−CH2), 41.91 (CH−CH2),
43.06 (CH2−NH−CO−NH), 51.59 (CH−CH2), 117.73 (C
N), 126.77 (C-4), 127.13 (C-2), 128.39 (C-3), 140.75 (C-1),
H
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given in Table S5. The quality of the final model was validated
with Molprobity.59 Atomic coordinates and structure factors
have been deposited in the Protein Data Bank with the
accession code 6YI7. The structure was analyzed using the
program CONTACT.54 All figures showing structural
presentations were prepared with the program PyMOL 1.4
for QM and SQM, respectively. Residues further than 6 Å of
3a in the crystal structure were frozen during the optimization.
To generate the noncovalent complexes of 3a and 3b with
SmCB1, it was necessary to break the covalent bond between
the inhibitor and the SG atom of Cys100 and transfer the
hydrogen atom of the CN−H group of the inhibitor to
His270.35 The bond was broken by employing harmonic
restraints and relaxed scans. The obtained noncovalent
complexes were reoptimized without any restraint and scored
using the standard QM-based scoring function.35,65,66 The
relative “free” energy of the noncovalent complex was
computed as a sum of gas phase interaction energy (ΔEint),
interaction desolvation free energy (ΔΔGsolv), and the change
of the conformational “free” energy of the ligand
(ΔGc′onf).35,65,66 The ligand structures were optimized in
solution by the same methodology as the complex. The
solvation free energy of the studied ligands was computed by
the SMD/HF/6-31G* method67 implemented in Gaus-
sian09.68
Life Cycle of Schistosoma mansoni. S. mansoni (NMRI
strain) is routinely maintained in the CDIPD by cycling
between Biomphalaria glabrata snails and Golden Syrian
hamsters.26,69,70 Vertebrate animal use is supported under a
protocol approved by UC San Diego’s Institutional Animal
Care and Use Committee (IACUC). The protocol complies
with United States federal regulations regarding the care and
use of laboratory animals: Public Law 99-158, the Health
Research Extension Act, and Public Law 99-198, the Animal
Welfare Act, which is regulated by USDA, APHIS, CFR, Title
9, Parts 1, 2, and 3. Informed consent of all participating
subjects was obtained.
Schistosome Phenotypic Assay. Newly transformed
schistosomula (NTS) of S. mansoni were prepared by
mechanically transforming infective larvae (cercariae) as
described previously.70,71 NTS (200−300 parasites) were
incubated in 200 μL of Basch Medium 169 containing 5%
FBS, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin at
5% CO2 and 37 °C.70,72,73 Inhibitors were added at the final
concentration of 10 μM,20,69 and changes in phenotypes were
observed every 24 h for up to 4 days. A constrained
nomenclature of “descriptors” is used to record the multiple
and dynamic changes in movement, shape, and translucence of
which the schistosome parasite is capable (Table S3).70 These
descriptors are then converted into an ordinal “severity score”
system from 0 (no effect) to 4 (maximum effect), which allows
for the relative comparison of compound effects, as
described.74,75 Images of NTS were captured using a Zeiss
Axiovert 40 C inverted microscope (10× objective) and a Zeiss
AxioCam MRc digital camera controlled by AxioVision 40 (v.
4.8.1.0) software.
̈
(Schrodinger, New York, USA).
Inhibition Assays. Inhibition measurements were per-
formed in triplicates in 96-well microplates (100 μL assay
volume) at 37 °C. SmCB1 (20−40 pM) was added to a
mixture of the fluorogenic substrate Cbz-Phe-Arg-AMC (20
μM) and an inhibitor (0−100 μM) in 0.1 M sodium acetate,
pH 5.5, containing 2.5 mM dithiothreitol and 0.1% PEG 6000.
The substrate hydrolysis was monitored in an Infinite M1000
̈
microplate reader (Tecan, Mannedorf, Switzerland) at
excitation and emission wavelengths of 360 and 465 nm,
respectively, for up to 15 and 60 min for fast- and slow-binding
inhibitors, respectively. Fast-binding inhibitors showed linear
progress curves, and the apparent inhibition constant Ki′ was
determined by nonlinear regression using the equation vs/v0 =
1/(1 + [I]/Ki′) by the GraFit software (Erithacus Software,
East Grinstead, UK), where vs is the steady-state velocity, v0 is
the velocity in the absence of an inhibitor, and [I] is the
inhibitor concentration. For slow-binding inhibitors, an
observed first-order rate constant kobs was calculated at each
inhibitor concentration by fitting the progress curve to the
equation P = vst + (vi − vs)(1 − exp(kobst))/kobs + d, where P is
the product formation, vs is the steady-state velocity, t is the
reaction time, vi is the initial velocity, and d is the offset. The
inhibition constant Ki′ was determined by nonlinear regression
using the equation vs/v0 = 1/(1 + [I]/Ki′). The true inhibition
constants Ki were then calculated using the equation Ki = Ki′/
(1 + [S]/Km), where [S] is the substrate concentration and Km
is the Michaelis constant. The apparent second-order rate
constant k2nd′ was determined by fitting to the linear equation
kobs = k2nd′[I] + koff, where koff is the first-order rate constants
for the dissociation of the enzyme−inhibitor complex and the
true constant k2nd was calculated by the correction k2nd
=
k2nd′(1 + [S]/Km). The Km value determined for Cbz-Phe-Arg-
AMC and SmCB1 was 25 μM. In none of the assay systems
did the final concentration of DMSO exceed 1.5%.
Interaction Energy Calculations. The crystal structure of
SmCB1 in complex with 3a was used for molecular modeling.
Hydrogen atoms were added to the protein by the Reduce and
Leap programs in AMBER 1460 and to the ligand using
PyMOL 1.7.6. Aspartates, glutamates, lysines, arginines, and
histidines were charged (except for the neutral His270 in
covalent complexes). Hydrogen atoms were relaxed by
annealing from 1000 to 0 K at the MM level in AMBER 14.
The FF14SB force field was used for the protein while the
GAFF force field was used for the ligand. The cooling runs
were 4 ps long with a 1 fs step and Berendsen thermostat used.
Fourteen crystallographic water molecules were considered for
the modeled complex. The 3c ligand was built manually using
the PyMOL 1.7.6.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00644.
The QM part comprised residues within 2.5 Å of 3a (i.e.,
300 atoms). The QM part was treated at the DFT-D3/BLYP/
TZVPP level for single-point energy calculations. For gradient
optimizations, we used the DFT-D3/BLYP/DZVP level.61 The
rest of the system was treated at the PM6-D3H4X level.62,63
The environment was described by the COSMO implicit
solvent model.56 The coupling between QM and SQM was
done by Cuby4,58 which calls Turbomole 7.057 and Mopac64
Supplementary methods, results, and six tables: (S1)
inhibition of SmCB1 by azadipeptide nitriles, (S2)
inhibition of SmCB1 by carbadipeptide nitriles, (S3)
antischistosomal activity of azanitrile and carbanitrile
inhibitors, (S4) cytotoxicity of the most potent azanitrile
inhibitors of SmCB1, (S5) X-ray data collection and
refinement statistics, and (S6) energy calculation of the
I
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https://pubs.acs.org/10.1021/acsinfecdis.0c00644
binding reaction of the inhibitors 3a and 3c to SmCB1;
four figures: (S1) atom labeling scheme for the inhibitor
3a, (S2) electron density maps for the inhibitor 3a, (S3)
energy calculation of the conformation change of the
inhibitors 3a and 3c, and (S4) the molecular surface of
the electrostatic potential (ESP) of the inhibitors 3a and
3c (PDF)
Author Contributions
‡A.J. and M.H. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Accession Codes
̌
́
̌
The authors thank Brian M. Suzuki, Jirı Brynda, and Vojtech
Spiwok for technical assistance with the phenotypic assays
involving the schistosome parasite, the crystallography, and the
statistics analysis, respectively. We also thank Helena
Atomic coordinates and experimental structure factors for the
SmCB1−3a complex have been deposited in the Protein Data
Bank with accession code 6YI7.
́
́
́
Mertlıkova-Kaiserova for cytotoxicity data. This work was
supported by grant LTAUSA19109 from the Ministry of
Education, Youth and Sports of the Czech Republic (MEYS),
grant NV18-05-00345 from the Ministry of Health of the
Czech Republic, project ChemBioDrug CZ.02.1.01/0.0/0.0/
16_019/0000729 from ERDF/OPRDE, Gilead Sciences &
IOCB Research Center, and institutional projects RVO
61388963 and PROGRES Q25. J.F. and M.L. were supported
by project LM2015070 from MEYS. Phenotypic screening at
the CDIPD was supported in part by R21AI126296 from NIH-
NIAID and OPP1171488 from the Bill and Melinda Gates
Foundation. Diffraction data were collected on MX14.1 at the
BESSY II electron storage ring operated by the Helmholtz-
Zentrum Berlin.
AUTHOR INFORMATION
Corresponding Authors
■
̌
Michael Mares − Institute of Organic Chemistry and
Biochemistry of the Czech Academy of Sciences, 16610
Prague 6, Czech Republic; Email: mares@uochb.cas.cz
Michael Gutschow − Pharmaceutical Institute,
̈
Pharmaceutical & Medicinal Chemistry, University of Bonn,
53121 Bonn, Germany; orcid.org/0000-0002-9376-
7897; Email: guetschow@uni-bonn.de
Authors
́
́
Adela Jílkova − Institute of Organic Chemistry and
Biochemistry of the Czech Academy of Sciences, 16610
Prague 6, Czech Republic; orcid.org/0000-0002-0847-
5022
ABBREVIATIONS
■
Martin Horn − Institute of Organic Chemistry and
Biochemistry of the Czech Academy of Sciences, 16610
Prague 6, Czech Republic; orcid.org/0000-0001-9110-
2018
AMC, aminomethylcoumarin; Bn, benzyl; Cbz, benzyloxycar-
bonyl; Cha, cyclohexylalanine; Hph, homophenylalanine;
NTS, newly transformed schistosomula; RMSD, root-mean-
square deviation; SmCB1, cathepsin B1 from Schistosoma
mansoni
̌
Jindrich Fanfrlík − Institute of Organic Chemistry and
Biochemistry of the Czech Academy of Sciences, 16610
Prague 6, Czech Republic
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̈
■
Medicinal Chemistry, University of Bonn, 53121 Bonn,
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